Over 150 mutations in the human rod opsin gene (hRHO) cause autosomal dominant retinitis pigmentosa (adRP). The folded opsin mRNA is the target for mutation-independent hammerhead ribozyme (hhRz) gene therapy. We have novel potent Facilitator-hhRz (F-hhRz) candidate therapeutics to treat all known hRHO mutations. The short-range goal is to translate effective F-hhRz therapeutics for hRHO adRP into human clinical trials. The objective is a preclinical F-hhRz proof-of-materials in mouse models of adRP that are ?humanized? for RHO mRNAs (same targets as in clinical trials). The central hypothesis is that reduction/ablation of toxic mutant hRHO mRNA/protein (P23H, P347S) will reduce stresses and slow/stop photoreceptor degeneration. The rationale is that Knockdown (KD) of the P23H or P347S hRHO mRNA/protein by a mutation-independent F-hhRz (also reduces wild type (WT) hRHO mRNA/protein) must be combined with Reconstitution (RECON) of WT hRHO protein expression (through an engineered ?non- cleavable? WT hRHO mRNA) to maintain photoreceptor vitality and function. To test the central hypothesis and accomplish the objectives the Specific Aims are: Aim 1. Screen for new Facilitator elements, using simple bioinformatics tools, that enhance cleavage rates of F-hhRzs at lead hRHO target sites (725 GUC?, 266 CUC?, 1362 GUC?). Expected results: F-hhRzs already perform >2 log units faster (150/min) than classical hhRzs (1-2/min). If protein enzyme rates (?1,000/min) are reached, even lower levels of in vivo F-hhRz expression will achieve equivalent KD. Aim 2. To test F-hhRz gene therapy agents for (i) rescue (efficacy) of retinal degeneration and (ii) toxicity, we conduct preclinical tests of the Solo-KD strategy with AAV-packaged F-hhRz gene therapy agents in simple transgenic mouse models. F-hhRz genes are delivered by state-of-art adeno-associated virus (AAV) vectors through subretinal or intravitreal injections. Different humanized adRP mouse models have different rates of outer retinal degeneration related to expression levels of mutant transgenes (robust evaluation of KD therapies) (on mouse WT RHO background), and the hWT model has no retinal degeneration. Expected results: rescue in adRP models depends only on potency (KD) by F-hhRz on mutant hRHO mRNA levels; F-hhRz KD related toxicity in the hWT model dictates minimum RECON level needed. Aim 3. To test lead F-hhRz agent and lead hardened WT hRHO construct for rescue of retinal degeneration in a fully humanized adRP model, we conduct a Combined AAV KD-RECON gene therapy strategy. Expected results: rescue depends on both sufficient toxic mutant protein KD and adequate WT RECON. The plan is innovative with a novel F-hhRz design, simple tools to improve Facilitator elements, novel humanized-for-target mouse models to simulate human trial, and novel approaches to harden WT hRHO mRNA for combined therapy. Significance- combined KD/RECON rescue in various fully humanized adRP models are robust efficacy outcomes to support clinical translation.